Innate immunity is broadly defined as the first line of defense against invading pathogens. Innate immune recognition is classically mediated by the interaction of pattern-recognition receptors (PRRs) and pathogen-associated molecular patterns (PAMPs), triggering serial downstream signaling events; alternatively, a number of enzymes such as lysozyme, digestive enzymes and defensins indiscriminately kill bacteria when the enzyme encounters the microbes. In the latter case, the enzyme does not directly recognize microbes, and the elimination of invading microbes is non-specific. Whether there is a PRR to directly kill invading microorganisms remains to be elucidated. Vascular peroxidase 1 (VPO1) is a newly-discovered mammalian heme-containing peroxidase (hPx). VPO1 is unique among the members of hPx family in that it contains a catalytic domain at its C-terminus and a large N-terminal region including five leucine-rich regions (LRRs) and four immunoglobulin (Ig) C2 type domains. VPO1 is highly expressed in the cardiovascular system, lung, liver, pancreas and spleen, and is secreted into bloodstream at a 1000-fold higher concentration than is MPO. However, its biological function has not been established. The central hypothesis of this proposed research is that VPO1 can recognize and directly kill invading microbes. Our specific aims are to (1) determine whether the LRR and Ig C2 domains of VPO1 bind to PAMPs; (2) determine if the binding of VPO1, via LRR and Ig C2, mediates microbicidal activity via generation of hypohalous acids. The major methods for addressing the aims include molecular cloning and expressing, protein purification, fluorescence polarization technology and surface plasmon resonance technology. Successful completion of this proposal will: (1) provide novel insights into innate immune responses; (2) identify the first mammalian protein with dual functions of pathogen recognition and killing; (3) create a new paradigm in the maintenance of bloodstream sterility by the physiological action of a novel dual function hPx; (4) elucidate the molecular mechanisms of VPO1-mediated pathogen recognition and killing, setting the foundation for the future development of diagnostics and/or therapeutics involving this novel innate immune pathway. PUBLIC HEALTH RELEVANCE: This project seeks is to identify and characterize a new mechanism of innate immunity in that both pathogen-recognition and killing occur within a heme-containing peroxidase, expanding our current concepts of innate immunity and filling the gap in innate immune system. The characterization of this activity and elucidation of this arm of the innate immune response will lead to potential new targets for therapies against infectious diseases.